Project Summary/Abstract A key driver of cancer is the global deregulation of transcription networks in the cell. Transcriptional output is dependent on chromatin structure and topology; repressed genes are sequestered from active genes into regions of compacted DNA near the nuclear membrane. Dense chromosomal domains, termed heterochromatin, are associated with Heterochromatin Protein-1 (HP1) and are tethered to the periphery via an interaction with the nuclear transmembrane protein, Lamin Binding Receptor (LBR), and the intermediate filament protein, Lamin B. Consequently, cancer cells that undergo global rewiring of transcription also often exhibit aberrant protein expression of Lamin B, LBR and HP1. The physical manifestation of the misregulation of these proteins is distortion in the size and shape of the nuclear envelope. Indeed, this nuclear pleomorphism is used as a histological marker of tumor progression. In estrogen receptor positive (ER+) breast cancer patients, downregulation of HP1 and upregulation of Lamin B and LBR is strongly correlated with earlier occurrence of distal metastasis. Understanding the biophysical properties that govern the assembly of heterochromatin at the nuclear periphery will facilitate the development of therapies aimed at restoring proper gene regulation. HP1 was recently found to concentrate DNA and chromatin into liquid-liquid phase separated (LLPS) droplets in vitro. This suggests a potential mechanism of DNA organization in vivo. To investigate the molecular details of HP1- mediated compaction and phase separation, I utilized DNA curtains and confocal microscopy. I identified key regions of HP1 required for multivalent?LLPS interactions and DNA compaction. This preliminary research focused on DNA, and the graduate work in Aim 1 will build on these studies by evaluating HP1 interactions with complex nucleosomal substrates. Heterochromatin in vivo is distinguished by evenly spaced nucleosomes and the trimethylation of histone H3 lysine 9 (H3K9me3). I will make chromatin substrates that range from mono- nucleosomes to 50kb chromatin fibers with variations in spacing and methylation modification. I will monitor the binding, oligomerization and phase separation of HP1 on these substrates by a combination of bulk biochemical assays and a novel single molecule chromatin assay. The proposed postdoctoral research in Aim 2 will focus on determining a physical model of the nuclear periphery. I will reconstitute the interactions between chromatin and the lamina with single molecule studies in vitro and super resolution studies in cells. I will use cell lines and mouse models of breast cancer metastasis to determine the molecular mechanism guiding metastatic recurrence of ER+ breast cancer patients with high expression of Lamin B and LBR. This research program will propel me toward my ultimate goal of leading my own lab studying how nuclear topology is coupled to cell fate determination in development, and the misregulation that leads to disease.